Alterations in Monocyte Phenotypes and Functions after a Hip Fracture in Elderly Individuals: A 6-Month Longitudinal Study.

BACKGROUND: Healthy elderly individuals are particularly prone to catastrophic events at any moment of their lives. One stressful event for individuals aged 65 and older is a fall that results in a fracture of the hip (HF). HF causes a state of inflammation that may affect immune responses. In this connection, we have reported that HF induced alterations in neutrophil functions. OBJECTIVE: To assess the impact of HF on classical (cM), intermediate (iM) and non-classical (ncM) monocyte subsets. METHODS: Distribution, functions (chemotaxis, phagocytosis, superoxide production and cytokine production), phenotype and activation (NF-x03BA;B and PI3K) were evaluated in monocyte subsets before surgery and 6 weeks and 6 months after the event. RESULTS: The distribution of cM and ncM was unchanged, but iM transiently increased before surgery. Sustained increases (iM response to CCL2 and CX3CL1) and decreases (cM and ncM response to CCL2) in chemotaxis were observed. Phagocytosis and superoxide production were impaired in cM but not in iM or ncM. Sustained expression of HLA-DR occurred in cM but not in iM and ncM. Sustained decreased expression of CD11b occurred only in ncM. Sustained decreases (cM and ncM) and increases (iM) in CCR2 expression were observed. An elevated expression of CX3CR1 was found only in iM. cM produced elevated quantities of TNFα. There was a transient oxidative burst of production before surgery in iM and a sustained decrease in ncM. IL-10 production was severely impaired in cM and decreased in iM prior to surgery. Sustained activation (cM), inhibition (ncM) and transient activation (iM) of NF-x03BA;B were observed. Activation of PI3K was severely impaired in cM and ncM but was sustained in iM. CONCLUSION: HF had more impact on cM and ncM functions than on iM. HF triggered a switch in cM functions from phagocytic to inflammatory elevated TNFα-producing cells. These changes may impact clinical outcomes of HF with respect to inflammation, opportunistic infections and physical recovery.

ß-Amyloid peptides display protective activity against the human Alzheimer's disease-associated herpes simplex virus-1.

Amyloid plaques, the hallmark of Alzheimer's disease (AD), contain fibrillar ß-amyloid (Aß) 1-40 and 1-42 peptides. Herpes simplex virus 1 (HSV-1) has been implicated as a risk factor for AD and found to co-localize within amyloid plaques. Aß 1-40 and Aß 1-42 display anti-bacterial, anti-yeast and anti-viral activities. Here, fibroblast, epithelial and neuronal cell lines were exposed to Aß 1-40 or Aß 1-42 and challenged with HSV-1. Quantitative analysis revealed that Aß 1-40 and Aß 1-42 inhibited HSV-1 replication when added 2 h prior to or concomitantly with virus challenge, but not when added 2 or 6 h after virus addition. In contrast, Aß 1-40 and Aß 1-42 did not prevent replication of the non-enveloped human adenovirus. In comparison, antimicrobial peptide LL-37 prevented HSV-1 infection independently of its sequence of addition. Our findings showed also that Aß 1-40 and Aß 1-42 acted directly on HSV-1 in a cell-free system and prevented viral entry into cells. The sequence homology between Aß and a proximal transmembrane region of HSV-1 glycoprotein B suggested that Aß interference with HSV-1 replication could involve its insertion into the HSV-1 envelope. Our data suggest that Aß peptides represent a novel class of antimicrobial peptides that protect against neurotropic enveloped virus infections such as HSV-1. Overproduction of Aß peptide to protect against latent herpes viruses and eventually against other infections, may contribute to amyloid plaque formation, and partially explain why brain infections play a pathogenic role in the progression of the sporadic form of AD.

Downregulation of inhibitory SRC homology 2 domain-containing phosphatase-1 (SHP-1) leads to recovery of T cell responses in elderly.

BACKGROUND: Immune responses are generally impaired in aged mammals. T cells have been extensively studied in this context due to the initial discovery of their reduced proliferative capacity with aging. The decreased responses involve altered signaling events associated with the early steps of T cell activation. The underlying causes of these changes are not fully understood but point to alterations in assembly of the machinery for T cell activation. Here, we have tested the hypothesis that the T cell pool in elderly subjects displayed reduced functional capacities due to altered negative feedback mechanisms that participate in the regulation of the early steps of T cell activation. Such conditions tip the immune balance in favor of altered T cell activation and a related decreased response in aging. RESULTS: We present evidence that the tyrosine phosphatase SHP-1, a key regulator of T cell signal transduction machinery is, at least in part, responsible for the impaired T cell activation in aging. We used tyrosine-specific mAbs and Western blot analysis to show that a deregulation of the Csk/PAG loop in activated T cells from elderly individuals favored the inactive form of tyrosine-phosphorylated Lck (Y505). Confocal microscopy analysis revealed that the dynamic movements of these regulatory proteins in lipid raft microdomains was altered in T cells of aged individuals. Enzymic assays showed that SHP-1 activity was upregulated in T cells of aged donors, in contrast to young subjects. Pharmacological inhibition of SHP-1 resulted in recovery of TCR/CD28-dependent lymphocyte proliferation and IL-2 production of aged individuals to levels approaching those of young donors. Significant differences in the active (Y394) and inactive (Y505) phosphorylation sites of Lck in response to T cell activation were observed in elderly donors as compared to young subjects, independently of CD45 isoform expression. CONCLUSIONS: Our data suggest that the role of SHP-1 in T cell activation extends to its increased effect in negative feedback in aging. Modulation of SHP-1 activity could be a target to restore altered T cell functions in aging. These observations could have far reaching consequences for improvement of immunosenescence and its clinical consequences such as infections, altered response to vaccination.

Stimulation of ENaC activity by rosiglitazone is PPARγ-dependent and correlates with SGK1 expression increase.

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists used to treat type 2 diabetes. TZD treatment induces side effects such as peripheral fluid retention, often leading to discontinuation of therapy. Previous studies have shown that PPARγ activation by TZD enhances the expression or function of the epithelial sodium channel (ENaC) through different mechanisms. However, the effect of TZDs on ENaC activity is not clearly understood. Here, we show that treating Xenopus laevis oocytes expressing ENaC and PPARγ with the TZD rosiglitazone (RGZ) produced a twofold increase of amiloride-sensitive sodium current (Iam), as measured by two-electrode voltage clamp. RGZ-induced ENaC activation was PPARγ-dependent since the PPARγ antagonist GW9662 blocked the activation. The RGZ-induced Iam increase was not mediated through direct serum- and glucocorticoid-regulated kinase (SGK1)-dependent phosphorylation of serine residue 594 on the human ENaC α-subunit but by the diminution of ENaC ubiquitination through the SGK1/Nedd4-2 pathway. In accordance, RGZ increased the activity of ENaC by enhancing its cell surface expression, most probably indirectly mediated through the increase of SGK1 expression.

E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cells.

The generation of knock-out mice for E2F4 gene expression has suggested a role for this transcription factor in establishing and/or maintaining the intestinal crypt compartment. Having previously demonstrated that E2F4 is cytoplasmic in quiescent-differentiated cells but nuclear in growth factor-stimulated proliferative cells, the present study was aimed at determining the role of E2F4 in the control of human intestinal epithelial proliferation. Results herein demonstrate that lentiviral infection of an shRNA which specifically knocked-down E2F4 expression slowed down G1/S phase transition and the proliferation rate of normal human intestinal epithelial cells (HIEC) and of colon cancer cells. Protein expression of Cdk2, cyclins D1 and A, Cdc25A and c-myc was markedly down-regulated in shE2F4-expressing cells; by contrast, expression of the cell cycle inhibitors p21(Cip/Waf) and p27(Kip1) was increased. In addition, the expression of many genes involved in DNA synthesis was down-regulated in shE2F4-expressing cells, whereas no modulation in E2F1 expression was observed. A decrease in E2F4 in colon cancer cell lines also resulted in a reduction in soft-agar growth capacity. Immunofluorescence experiments in human fetal intestine revealed that cells expressing high nuclear levels of E2F4 also expressed cyclin A protein. Lastly, E2F4 and its target cyclin A were up-regulated and mostly nuclear in human colorectal tumor cells in comparison to the corresponding benign epithelium. These results indicate that nuclear E2F4 may be determinant in the promotion of proliferation of human intestinal epithelial crypt cells and colorectal cancer cells.

Cytomegalovirus Seropositivity Predicts a Decline in the T Cell But Not the Antibody Response to Influenza in Vaccinated Older Adults Independent of Type 2 Diabetes Status.

Type 2 diabetes mellitus (T2DM) and persistent cytomegalovirus (CMV) infection are postulated contributors to inflammatory processes that impact on the age-related decline in T-cell responses to influenza vaccination. Older subjects with T2DM (n = 30) and healthy aged controls (n = 40) were enrolled and received influenza vaccination in this study. Serum inflammatory markers and CMV serostatus were measured. Pre- to post-vaccination changes in serum antibody titers to the A/H3N2 strain, and levels of granzyme B (GrB, cytotoxic T lymphocytes) in lysates and cytokines in supernatants from influenza A/H3N2-challenged peripheral blood mononuclear cells were measured. We found no difference between the T2DM and healthy groups in the immune responses measured. However, CMV serostatus was a key determinant of the GrB response to influenza challenge; CMV+ subjects had low levels of inducible GrB (iGrB) activity in response to influenza challenge. In contrast, the serum antibody response to the A/H3N2 vaccine strain did not differ with CMV serostatus, and serum levels of the inflammatory marker, ß2-microglobulin, were positively correlated with age, T2DM, and serum IL-10 levels. In conclusion, CMV seropositivity associated with a decline in GrB responses to influenza may predict increased susceptibility to influenza in older adults.

Differential Phenotypes of Myeloid-Derived Suppressor and T Regulatory Cells and Cytokine Levels in Amnestic Mild Cognitive Impairment Subjects Compared to Mild Alzheimer Diseased Patients.

Alzheimer disease (AD) is the most prevalent form of dementia although the underlying cause(s) remains unknown at this time. However, neuroinflammation is believed to play an important role and suspected contributing immune parameters can be revealed in studies comparing patients at the stage of amnestic mild cognitive impairment (aMCI) to healthy age-matched individuals. A network of immune regulatory cells including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) maintains immune homeostasis but there are very few data on the role of these cells in AD. Here, we investigated the presence of these cells in the blood of subjects with aMCI and mild AD (mAD) in comparison with healthy age-matched controls. We also quantitated several pro- and anti-inflammatory cytokines in sera which can influence the development and activation of these cells. We found significantly higher levels of MDSCs and Tregs in aMCI but not in mAD patients, as well as higher serum IL-1ß levels. Stratifying the subjects based on CMV serostatus that is known to influence multiple immune parameters showed an absence of differences between aMCI subjects compared to mAD patients and healthy controls. We suggest that the increase in MDSCs and Tregs number in aMCI subjects may have a beneficial role in modulating inflammatory processes. However, this protective mechanism may have failed in mAD patients, allowing progression of the disease. This working hypothesis obviously requires testing in future studies.

Predictors of the antibody response to influenza vaccination in older adults with type 2 diabetes.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic inflammatory diseases of the elderly. Its development is related to the alteration of the immune system with aging characterized by immunosenescence and inflamm-aging. In turn, T2DM also alters the immune response. As a consequence, older people with T2DM are more susceptible to influenza and to its complications as compared with healthy controls. Vaccination against influenza has shown poor efficacy in the older population and even less efficacy in patients with diabetes. We studied here the antibody response to vaccination in healthy and diabetic elderly participants. RESEARCH DESIGN AND METHODS: In 2 groups of elderly participants (healthy N=119 and T2DM N=102), we measured the immunogenicity of influenza vaccine by hemagglutination inhibition assays. We assessed several blood and functional parameters as potential predictors of the vaccine efficacy. RESULTS: We found no difference between antibody responses in diabetic elderly compared with healthy elderly. Among the biological and functional determinants, the cytomegalovirus (CMV) serostatus played a more prominent role in determining the magnitude of response. We concluded that in addition to age and diabetic status, immunological history such as CMV status should be taken into account. None of the other biological or functional parameters studied could be reliably linked to the vaccine antibody response in older adults who are not frail including those with well-controlled diabetes. CONCLUSIONS: Our data strongly suggest that influenza vaccine should be administered to elderly patients with T2DM; however, the immune determinants of the antibody response to influenza vaccination should be further investigated.

Altered neutrophil functions in elderly patients during a 6-month follow-up period after a hip fracture.

BACKGROUND: Fracture of the hip (HF) is a significant cause of morbidity and mortality in elderly individuals. HF is an acute stress that triggers a state of inflammation which may affect immune responses and physical recovery. METHODS: Longitudinal study of the impact of HF on the functions of polymorphonuclear neutrophils (PMNs) in elderly subjects. Data were recorded prior to surgery, 6weeks and 6months later. RESULTS: PMN functions were severely impaired shortly after HF (chemotaxis, phagocytosis, superoxide production) but there was a time-related recovery of some PMN functions (chemotaxis, phagocytosis) over time, except in the case of superoxide production. Whereas FcγRII (CD32) expression remained unchanged, FcγRIII (CD16) increased from low values before surgery to levels of controls 6months post-surgery. This was also the case for the C5a complement receptor and CD11b. TLR2 and TLR4 expressions were unchanged. Cytokine and chemokine secretions by stimulated PMN were altered. TNFα and IL-10 secretions were increased following HF but IL-8 secretion was decreased. Impaired PMN functions prior to surgery were related to alterations in PI3K and NF-κB signaling pathways. Recovery of these functions paralleled increased PI3K activity, although superoxide production remained low. Sustained activation of the NF-κB pathway by TNFα has been reported to involve upregulation of IKKß kinase activity. Activated IKKß kinase inhibits ERK1/2 and results in concomitant downstream inhibition of NADPH oxidase complex which can account for sustained impaired production of ROS in HF patients. CONCLUSION: Our data showed that the stress caused by HF negatively affects initial PMN responses shortly after the event and that may negatively influence clinical outcomes such as resolving long-term inflammation and recovery, as well as explaining susceptibility to opportunistic infections.

Aging, immunosenescence and membrane rafts: the lipid connection.

The decreased efficiency of immune responses in older people is partly a consequence of alterations in T lymphocyte functions caused by modifications in the early events of signal transduction. Several alterations in the signaling pathways of T lymphocytes have been described in older humans and animals. A unifying cause could be modifications in the physicochemical properties of the plasma membrane resulting from changes in its lipid composition and the distribution and function of lipid rafts (LR). The latter serve to assemble the initial components of the signaling cascade. Accumulating data suggest that the function of plasma membrane LR is altered with aging; we hypothesize that this would significantly contribute to immune dysregulation. The role of aging and cholesterol in LR functions in T lymphocytes is reviewed and discussed here.

Nuclear expression of E2F4 induces cell death via multiple pathways in normal human intestinal epithelial crypt cells but not in colon cancer cells.

E2F transcription factors control cell cycle progression. The localization of E2F4 in intestinal epithelial cells is cell cycle dependent, being cytoplasmic in quiescent differentiated cells but nuclear in proliferative cells. However, whether nuclear translocation of E2F4 alone is sufficient to trigger intestinal epithelial cell proliferation remains to be established. Adenoviruses expressing fusion proteins between green fluorescent protein (GFP) and wild-type (wt)E2F4 or GFP and nuclear localization signal (NLS)-tagged E2F4 were used to infect normal human intestinal epithelial crypt cells (HIEC). In contrast to expression of wtE2F4, persistent expression of E2F4 into the nucleus of HIEC triggered phosphatidylserine exposure, cytoplasmic shrinkage, zeiosis, formation of apoptotic bodies, and activation of caspase 9 and caspase 3. Inhibition of caspase activities by zVAD-fmk partially inhibited cell death induced by E2F4-NLS. An induction of p53, phosphorylated Ser15-p53, PUMA, FAS, BAX, RIP, and phosphorylated JNK1 was also observed in HIEC expressing E2F4-NLS compared with wtE2F4-expressing cells. E2F1 and p14ARF expression remained unaltered. Downregulation of p53 expression by RNA interference attenuated cell death induced by E2F4-NLS. By contrast, the level of cell death was negligible in colon cancer cells despite the strong expression of E2F4 into the nucleus. In conclusion, deregulated nuclear E2F4 expression induces apoptosis via multiple pathways in normal intestinal epithelial cells but not in colon cancer cells. Hence, mutations that deregulate E2F4 localization may provide an initial proliferative advantage but at the same time accelerate cell death. However, intestinal cells acquiring mutations (e.g., p53, Bax loci, etc.) may escape apoptosis, thereby revealing the full mitogenic potential of the E2F4 transcription factor.